Mesenchymal stem cells (MSCs) pose exciting possibilities for repairing tissues damaged by injury or disease. Their relative abundance and capability to become many different cell types make them attractive as an alternative autologous cell source. However, the cellular heterogeneity present in MSC harvests complicates their therapeutic application since not all cells will behave in the same manner. Individual stem cells respond to biochemical and mechanical stimuli in their local microenvironment, which can direct differentiation along specific pathways/lineages. While substrate compliance has been extensively investigated in recent years as it relates to stem cell differentiation, little attention has been given to how the mechanical properties of individual cells can influence this process for themselves and their neighbors. Findings from the PI?s laboratory indicate cellular mechanical properties correlate with biological characteristics, such as gene expressions, and play an important role in determining the differentiation fate of stem cells. Not all MSCs behave uniformly, and this heterogeneity can dramatically impact the overall response of a sample. Understanding how the biological and mechanical characteristics of local cell populations relate to the larger group can provide clues to optimizing future regenerative therapies.
The proposed project will investigate the heterogeneity of MSC samples at the single-cell, neighboring (~101 cells), and population (~103+ cells) levels using two complimentary strategies. First, live-cell, gene expression-based markers will be used to identify differentiating and non-differentiating MSCs that have been chemically induced for osteogenesis and adipogenesis. Second, single-cell, elastic and viscoelastic testing will assess the mechanical heterogeneity in undifferentiated, ?partially? differentiated, and fully differentiated stem cell cultures. In addition to expanding knowledge of stem cell mechanics, this research program has the potential to vastly improve experimental approaches that strive to direct stem cell differentiation and improve regenerative responses following implantation.
Intellectual Merit: The proposed project seeks to investigate the inherent heterogeneity present in mesenchymal stem cell populations. MSCs are typically investigated at the population level, which can obscure variations that exist among individual cells. Little is known about how single-cell, mechanical properties change during differentiation, but this knowledge is critical for fully understanding cell-substrate and cell-cell behavior. Likewise, elucidating the spatiotemporal patterns of gene expression in MSC samples can help determine optimal times and locations for biochemical stimulation. To pursue these goals, this project will use custom-designed molecular beacons to visualize mRNA molecules in live, differentiating MSCs. Atomic force microscopy will be used to mechanically characterize the elastic and viscoelastic properties of individual cells. Focus will be given to how local cell populations behave, biochemically and mechanically, in relation to the overall sample. Findings will be applicable to many fields, including stem cell biology, cell mechanics, and tissue engineering. The PI is a pioneer in the area of single-cell, mechanical biomarkers, an exciting new field that provides phenotypic characterization akin to gene and protein expression. The proposed research furthers the maturation of these transformative approaches by investigating the role of mechanical biomarkers in stem cell differentiation.
Broader Impact: As a biomedical engineer conducting research at the cross-section of math, biology, chemistry, and engineering, the PI is well-suited to provide an environment that highlights the need for interdisciplinary understanding. The outreach effort proposed here will provide primary and secondary school educators at the Providence Public School District the opportunity to conduct team-based research during the summer. Teachers specializing in different subjects will work together on projects that incorporate multiple areas of expertise. The goal of this project is to facilitate interaction among teachers so that they can develop interdisciplinary examples and experiments for their own classes. Students take many math and science courses throughout their education, encompassing algebra, geometry, biology, chemistry, and physics. However, these courses are almost always taught as isolated subjects, whereas real-world applications are increasingly interdisciplinary. The proposed project will be conducted as an extension of Brown University?s established GK-12 program. In addition to this outreach effort, the PI will refine his recently designed course on stem cell engineering for graduate and undergraduate students that covers areas of stem cell biology, regenerative medicine, and hands-on laboratory exercises. Experimental findings from the research portion of this proposal will be directly applicable to this course, which is the first of its kind at Brown University.
